The objective of the proposed research is to develop a better understanding of how enzymes activate stable covalent bonds. A mechanistic investigation of biologically and medically significant C-H bond activation by the enzyme dihydrofolate reductase (DHFR) will be conducted. We will examine the role of protein dynamics, coupled motion, and quantum mechanical hydrogen tunneling in catalysis. Tunneling is the phenomenon by which a particle transfers through a reaction energy barrier due to its wave-like property. Coupled motion of several nuclei along the reaction coordinate is another phenomenon that has been found to be important in some enzyme catalyzed reactions. The principal problem we wish to address is: does the enzyme's dynamics enhance the chemistry it catalyzes? We propose to investigate the effect of altered enzyme dynamics on the nature of the C-H-C transfer that it catalyzes. Tunneling and coupled motion will serve as probes for the nature of the chemical step in the enzyme complex catalytic cascade. The enzyme's dynamics will be altered by site-directed mutagenesis and other methods. The effect of the altered dynamics on the nature of the H-transfer will be examined. Several theoretical models will be applied to correlate the dynamics to the degree of tunneling and coupled motion. In the project proposed herein, a method will be developed to study the degree of tunneling and coupled motion in the DHFR catalyzed reaction. The experimental design includes: - Measuring the ratio of reaction rates with substrates labeled with the three isotopes of hydrogen (H/D/T kinetic isotope effects) and their temperature dependence. - Data analysis using non-classical methods, which will afford an estimation of the degree of tunneling and coupled motion in the hydride transfer. - Pursuing a correlation between the protein dynamics and the nature of H-transfer. The proposed project will lead to significant insight into the mechanism of DHFR and fundamental aspects of biocatalysis in general. A methodology for looking into hydride tunneling with a common biological reductive cofactor (nicotinamide) would be extremely useful for studying the many enzymes utilizing this cofactor. A better mechanistic understanding of these enzymes could lead to new approaches to the rational design of inhibitors and would facilitate the design of drugs.